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PubMed1996

Acute effects of morphine on Substance P concentrations in microdissected regions of guinea-pig brain.

The present study investigated the effects of acute morphine treatment and of naloxone-induced morphine withdrawal on Substance P (SP) concentrations in microdissected regions of the guinea-pig brain. Guinea-pigs, which were treated with a single dose of morphine sulphate (15mg/kg s.c.), received naloxone hydrochloride (15mg/kg s.c.) after 2h. Control animals received injections of saline, saline and naloxone, or morphine and saline. Locomotor and behavioural activities were measured throughout the experiments. Animals were killed 0.5h after naloxone administration, brains were removed and SP-like immunoreactivity (SP-LI) was measured in microdissected regions using radioimmunoassay. Morphine significantly increased the concentration of SP-LI in the central nucleus of the amygdala, but reduced SP-LI overall in the mesencephalon. Guinea-pigs pretreated with morphine and then given naloxone to precipitate withdrawal showed no change in SP-LI concentrations in any brain region, compared with control animals, despite an increase in locomotor activity and other behaviours characteristic of opioid withdrawal in guinea-pigs.

Journal Article↗
PubMedDate not supplied

The role of tissue microdissection in cancer research.

Tissue microdissection is a laboratory method that is used to procure specific cells or cell populations from a histology slide under direct microscopic visualization. The recovered cells can be studied with a variety of DNA, messenger RNA, and protein analysis methods, including new high-throughput gene expression and proteomics technologies. This approach is permitting investigators to comprehensivelyexamine the molecular anatomy of cells in tissue sections forthe first time. This article reviews the development and evolution of tissue microdissection techniques, summarizes examples of research studies, and discusses related challenges that the research community must address. Additional information and complete laboratory protocols are available on a website at http://cgap-mf.nih.gov/.

DNA, Neoplasm↗
PubMed2001

Identification and validation of P311 as a glioblastoma invasion gene using laser capture microdissection.

The mRNA expression profiles from glioblastoma cells residing at the tumor core and invasive rim of a human tumor resection were compared. From a single tumor specimen, 20,000 single cells from each region were collected by laser capture microdissection. Differential expression of 50-60 cDNA bands was detected. One of the sequences overexpressed by the invasive cells showed 99% homology to the P311 gene, the protein product of which is reported to localize at focal adhesions. Relative overexpression of P311 by invading glioblastoma cells compared with tumor core was confirmed by quantitative reverse transcription-PCR of six glioblastoma specimens after laser capture microdissection collection of rim and core cells. In vitro studies using antisense oligodeoxynucleotides and integrin activation confirmed the role of P311 in supporting migration of malignant glioma cells. Immunochemistry studies confirmed the presence of the P311 protein in tumor cells, particularly at the invasive edge of human glioblastoma specimens.

Amino Acid Sequence↗
PubMed2002

Analysis of microdissected prostate tissue with ProteinChip arrays--a way to new insights into carcinogenesis and to diagnostic tools.

Prostate carcinomas are one of the most common malignancies in western societies. The pathogenesis of this tumor is still poorly understood. These tumors present with two characteristic features: epithelial-mesenchymal interactions, which play a pivotal role for tumor development and most of clinically manifest cancers arise in prostate proper compared to a minority of tumors which develop in the transitional zone. Deciphering the epithelial-mesenchymal cross talk and identification of molecular pecularities of the sub-populations of cells in different zones can therefore help understanding carcinogenesis and development of new, non-invasive tools for the diagnosis and prognosis of prostate carcinomas which has remained a challenge until today. A ProteinChip array technology (SELDI = surface enhanced laser desorption ionization) has been developed recently by Ciphergen Biosystems enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the analysis of approximately 500-1000 freshly obtained prostate cells by SELDI-TOF-MS (surface enhanced laser desorption ionization time-of-flight mass spectrometry). Pure cell populations of stroma, epithelium and tumor cells were selected by laser assisted microdissection. Multiple specific protein patterns were reproducibly detected in the range from 1.5 to 30 kDa in 28 sub-populations of 4 tumorous prostates and 1 control. A specific 4.3 kDa peak was increased in the prostate tumor stroma compared to normal prostate proper and transitional zone stroma and increased in prostate tumor glands compared to normal prostate proper and transitional zone glands. Coupling laser assisted microdissection with SELDI provides tremendous opportunities to identify cell and tumor specific proteins to understand molecular events underlying prostate carcinoma development. It underlines the vast potential of this technology to better understand pathogenesis and identify potential candidates for new specific biomarkers in general which could help to screen for and distinguish disease entities, i.e. between clinically significant and insignificant carcinomas of the prostate.

Aged↗
PubMed2002

Identification of cyclin D1 mRNA overexpression in B-cell neoplasias by real-time reverse transcription-PCR of microdissected paraffin sections.

PURPOSE: Overexpression of cyclin D1 mRNA and protein as a result of the chromosomal translocation t(11;14)(q13;q32) is a highly specific molecular marker of mantle cell lymphoma, but cyclin D1 dysregulation can also be found in other B-cell neoplasias. The aim of the study was to develop a precise and reliable tool for quantitation of cyclin D1 mRNA suitable for archival clinical specimens. EXPERIMENTAL DESIGN: A real-time reverse transcription-PCR (RT-PCR) assay was used to quantitate cyclin D1 mRNA copy numbers. Using 2000 microdissected cells as template, 104 formalin-fixed, paraffin-embedded lymph node, spleen, and decalcified bone marrow biopsies from a panel of 95 cases of B-cell non-Hodgkin's lymphomas (B-NHLs) were analyzed. In addition, cyclin D1 protein expression was assessed by immunohistochemistry. RESULTS: Strong cyclin D1 mRNA overexpression was detected in mantle cell lymphomas (23 of 23), hairy cell leukemias (5 of 19), and multiple myelomas (7 of 23) with particularly high levels in 2 of the latter cases. Intermediate transcript levels were found in 5 of 23 multiple myelomas and 7 of 19 hairy cell leukemias. B-cell chronic lymphocytic leukemias (10 of 10), follicular lymphomas (9 of 9), mucosa-associated lymphoid tissue lymphomas (5 of 5) and reactive lymphoid tissues with the exception of normal spleen had no or very low cyclin D1 expression. In comparison with real-time RT-PCR, immunohistochemistry showed a lower level of sensitivity, more variability, and did not allow accurate quantitation. CONCLUSIONS: Real-time RT-PCR for cyclin D1 mRNA is an excellent tool for the differential diagnosis of B-NHLs and, in combination with microdissection, a powerful approach for retrospective trials using archival clinical specimens as tissue source. Furthermore, real-time RT-PCR may help to identify subgroups of B-NHLs according to cyclin D1 mRNA copy numbers and to investigate the possible influence of different chromosomal breakpoints on cyclin D1 expression.

Chromosomes, Human, Pair 11↗
PubMed2002

[Sharper diagnostic tool the future promise of laser-assisted microdissection].

The paper describes the use of laser-assisted microdissection to retrieve microscopically defined cell populations including single cells from tissue sections for subsequent analysis of genomic DNA and mRNA. A general background is given on the techniques available and requirements for PCR based on minute templates. Different pre-PCR approaches are briefly described and possibilities and limitations of using archival material compared to fresh frozen tissue are discussed. In the article we give one example on how we have used the PALM laser microscopy system in combination with a nested, multiplex PCR system to analyze single normal keratinocytes as well as tumor cells from a case of basal cell cancer. We found that p53 mutations are common in normal, chronically sun-exposed skin. Widespread yet common mutations in the p53 gene that were unrelated to immunoreactivity for the p53 antibody were found in tumor cells. In addition there were rare mutations in occasional tumor cells that apparently did not result in selective growth advantage. Perspectives for the future are presented and the potential of laser assisted microdissection is highlighted within the fields of cancer research, developmental studies as well as studies of inflammatory and degenerative diseases. The combination of a method that allows careful selection of defined cells with powerful micro array based techniques, provides a setting with potential to uncover pathogenic mechanisms for large variety of human diseases.

Cytological Techniques↗
PubMed2002

Loss of heterozygosity at microsatellite markers from region p11-21 of chromosome 8 in microdissected breast tumor but not in peritumoral cells.

Alterations of chromosomal region 8p11-21 are very frequent in human cancers, and especially in breast cancer; yet, most of the genes involved have not been identified. We performed laser capture microdissection in a series of 52 consecutive breast tumor samples to obtain pure tumor cells without surrounding normal breast. To determine genomic subregions in which some of the cancer genes may be located, we conducted a search for loss of heterozygosity (LOH) at 13 microsatellite markers from this region. Two-thirds of the tumors showed LOH at least at one marker. Microdissection of pure tumor samples was helpful to precisely define four LOH subregions. No LOH was observed in the corresponding peritumoral tissues. We studied by immunohistochemistry (IHC) on tissue-microarrays the expression in the same tumors, of the protein product of three potential tumor genes lying close to or within the subregions of LOH. In most samples, the TACC1 gene product was downregulated in tumor cells as compared to normal cells. Our results show that the centromeric portion of chromosome arm 8p is frequently altered in breast tumor cells.

Breast Neoplasms↗
PubMed2002

[Microdissection and PCR-SSCP detected mutation and expression of p53 and K-ras gene in carcinogenesis and development of induced rat lung carcinoma].

OBJECTIVE: To investigate the roles of p53 and K-ras gene in carcinogenesis and development of the lung carcinoma induced by 3-methylcholanthrene (MCA) and diethylinitrosamine (DEN) in Wistar rats, and to elucidate the relationships between the protein expression and gene mutation of p53 and K-ras. METHODS: Microdissection was used to obtain pure cell populations of each phase in the carcinogenesis and development of lung carcinoma induced by MCA and DEN. DNA of the microdissected cell populations was extracted and used to analyze the mutations of p53 exons 5 approximately 8 and K-ras exons 1 approximately 2 by PCR-SSCP. The expressions of p53 and K-ras protein in each phase were detected by immunohistochemistry. RESULTS: No mutation and protein expression of p53 and K-ras was found in the 30 cases with normal bronchial epithelium. Mutation of p53 was detected in 3.1% of 18 hyperplasia and 14 squamous metaplasia cases, 28.6% of 21 dysplasia, 30.0% of 12 carcinomas in situ, 51.2% of 43 infiltration carcinomas, 52.9% of 17 metastases. The positive immunostaining rate of p53 protein was 0, 42.9%, 50.0%, 60.5% and 64.7% respectively. K-ras mutation rate was 0, 4.8%, 8.3%, 9.3%, 11.8% respectively, while the overexpression rate of K-ras protein was 15.6%, 19.0%, 25.0%, 41.9%, 52.9% respectively. p53 protein expression was closely related with p53 mutation (P < 0.005, Pearson's R = 0.599 6). There was no relationship between the protein expression and gene mutation of K-ras (P > 0.500). CONCLUSIONS: p53 gene mutation and K-ras overexpression were early events in the carcinogenesis and development of rat lung carcinoma induced by MCA and DEN, while K-ras mutation does not play any important role.

Animals↗
PubMed1999

[Microdissection of M chromosome in Vicia faba and its library construction].

Microdissection and microcloning technique was employed to construct the library of M chromosome in Vicia faba. The M chromosomes were microdissected with a micromanipulator and were put into a 0.5 ml Eppendorf tube, then digested with Sau3A. Sau3A linker adaptors were ligated to the end of chromosome DNA fragments, and two rounds of PCR were carried out with one chain of linker adaptor as the primer. The PCR products ranged in size from 300 base pair (bp) to 3000 bp with predominant fragments from 500 bp to 1500 bp. Southern hybridization analysis confirmed that PCR products originated from Vicia faba genome. The second round PCR products were cloned and about 102,000 recombinants were obtained. 118 recombinants were selected randomly for analysis. The inserts ranged in size from 150 bp to 3000 bp with an average of 690 bp. Dot blot was carried out for 100 clones with DIG labeled Vicia faba genome DNA as probes. The result revealed that 51% were low and unique copy sequences, 49% were repetitive sequences. M chromosome DNA library has not been reported before.

Chromosome Mapping↗
PubMed2003

Loss of cellular retinol-binding protein 1 gene expression in microdissected human ovarian cancer.

PURPOSE: We have previously found that cellular retinol-binding protein 1 (CRBP1),involved in retinol transport and metabolism, is down-regulated in an in vitro rat model of ovarian cancer and in several human ovarian cancer cell lines. The aim of this study was to determine the clinical relevance of this change to human ovarian cancer. EXPERIMENTAL DESIGN: A cohort of 48 frozen human serous ovarian carcinomas was evaluated for CRBP1 gene expression. Malignant ovarian epithelial cells were selectively procured by laser capture microdissection, and their CRBP1 expression was determined by real-time PCR. Immunohistochemistry for CRBP1 was performed on paraffin sections of ovarian tumors using polyclonal affinity-purified rabbit anti-CRBP1 antibody. RESULTS: In 35% of ovarian cancer patient samples, there was no detectable CRBP1 expression by real-time PCR. The expression of CRBP1 in microdissected serous ovarian carcinomas was not related to either tumor stage (P = 0.6839) or grade (P = 0.9599). Quantitative PCR results were confirmed by immunohistochemistry using an antibody against CRBP1. CONCLUSIONS: The loss of CRBP1 expression in clinical ovarian tumor specimens is consistent with our previous findings in the rat model and human ovarian cancer cell lines. It appears to be an early event in ovarian carcinogenesis because there was no statistically significant difference in its frequency between tumor stages and grades. Our findings suggest that the loss of CRBP1 expression contributes to the ovarian cancer oncogenesis via altered vitamin A metabolism.

Activating Transcription Factor 2↗
PubMed1992

Frequency and significance of cervicomediastinal lymph node metastases in medullary thyroid carcinoma: results of a compartment-oriented microdissection method.

The frequency and significance of cervicomediastinal lymph node metastases have been investigated in 82 medullary thyroid carcinoma (MTC) patients retrospectively comparing two surgical techniques of lymph node dissection: selective lymphadenectomy (n = 63) versus compartment-oriented microdissection (n = 35). No positive correlation was observed between primary tumor size and the number of lymph node metastases. In patients with lymph node metastases proven histologically, 42% showed only cervical involvement (35% unilateral--type A, 7% bilateral--type B) and 22% cervicomediastinal lymph node involvement (15% cervico-unilateral and mediastinal--type C, 7% cervicobilateral and mediastinal--type D). Biochemical cure was 83% in node-negative patients but only 21% in node-positive patients. In node-positive MTC, calcitonin normalization was achieved in none with bilateral lymph node involvement but only in those unilateral lymph node metastases (31% in type A, 17% in type C). Survival and biochemical cure are significantly improved by application of the compartment-oriented microdissection method more so at primary surgery than at reoperation.

Carcinoma↗
PubMed1991

[Technique for the microdissection of human high resolution chromosome subsequent polymerase chain reaction and microcloning].

In this paper is reported a simple technique for the microdissection of specific regions of human high resolution chromosome subsequent PCR and microcloning. This technique was successfully used to microdissect 4 chromosomal pieces from the distal one third from band 11.2 to the terminal of the short arm of Y chromosome where the testis determining factor located, PCR 30 cycles, and obtained 3.6 x 10(4) clones. This technique the highlight to establish a gene library of many specific regions of human chromosome hereafter.

Cloning, Molecular↗
PubMed1991

Microdissection of human high resolution banded chromosome, polymerase chain reaction and microcloning.

A simple technique for microdissection of specific region of human high resolution banded chromosome, followed by polymerase chain reaction (PCR), and microcloning was successfully used to microdissect 4 chromosomal pieces from the distal one third from band 11.2 to the terminal of the short arm of Y chromosome where the testis determining factor is located; 3.6 x 10(4) clones were obtained after 30 cycles of PCR. We analysed 41 clones with insert. The size of insert ranges from 140 to 350 bp (average 250 bp). A Southern blot analysis was done for one of them, and a 2.5 kb Hind III fragment was detected.

Cloning, Molecular↗
PubMed1988

The gill arch of the striped bass, Morone saxatilis. III. Morphology of the basal lamina as revealed by various ultrasonic microdissection procedures.

The ultrastructure of the surface epithelium and the associated basal lamina of the gill arches of the striped bass, Morone saxatilis, were investigated with the scanning electron microscope following complete or partial removal of the epithelium by ultrasonic microdissection. The microdissection procedures employed various combinations of the techniques of aldehyde fixation, treatment with borate, and extensive osmication followed by mild sonication. Generally, aldehyde fixation increases intercellular adhesion, excess osmication increases tissues brittleness, and borate treatment causes extensive tissue dissociation. However, the degree of epithelial removal following sonication of tissues treated with these various procedures varies considerably with specimen structure, shape, proximity to adjacent structures and freedom to vibrate during sonication. The basal lamina exhibits a smooth contour over most of the gill surface with the exception of the short gill rakers where it formed cones within the taste bud cores, and on the respiratory lamellae where it closely mimicked the underlying capillary network.

Animals↗
PubMed1986

Ultrasonic microdissection of immature intermediate human placental villi as studied by scanning electron microscopy.

The human placenta during the first 20 weeks of gestation undergoes rapid and extensive morphological changes. Near the end of this period, the most predominant type of villus present is the immature intermediate placental villus. In order to visualize this complex structure with scanning electron microscopy (SEM), we have developed a microdissection technique to expose tissue components of the placental villus while retaining its normal histological architecture. Placental villi were initially fixed in Karnovsky's fixative, buffered formalin, or 2% osmium tetroxide solution prior to exposure to connective tissue enzymes or detergents alone or in combination. Samples were dehydrated through 100% acetone and ultrasonicated at 80 kHz for 15 minutes prior to critical point drying and SEM examination. The most satisfactory microdissections were obtained by using a combined detergent/ultrasonication technique. By means of this procedure it was possible to remove the syncytiotrophoblast to expose the underlying cytotrophoblast, basal lamina and the stromal core components of the villi. The selective removal of these structures revealed the 3-dimensional relationships of the stromal channels, reticulum cells and Hofbauer cells. Of interest was the pattern of fetal capillaries coursing parallel to the long axis of each villus and terminating in a vascular knot at the tip.

Cell Fractionation↗
PubMed1978

Cystic kidneys, renal dysplasia and microdissection data in 5 children with congenital valvular urethral obstruction.

A survey of the findings in 5 children with urethral obstruction due to valves is presented. The microdissection data found in the 7 kidneys of this series were compared. The microdissection findings in all 7 kidneys are mostly the same with a continuum of processes ranging from relatively mild to severe derangements. We could not find two varieties of renal change designated type II or IV by potter. The characterization of the lesions by nephron dissection always approached those of the type II (multicystic, dysplastic kidney). Renal dysplasia was associated with extrarenal malformations in 2 of our cases and we therefore believe that the significance of the dysplasia lies rather in the period of the insult during organogenesis than in the obstruction.

Autopsy↗
PubMed1978

Structural changes of experimental glomerulonephritis in rats as revealed by microdissection.

Microdissection revealed striking alterations in the nephrons of animal models AICN and anti-GBMN. The most severely damaged AICN kidney in the series presented marked heterogeneity in the size and deformity of the proximal tubules, corresponding to the diverse and variegated nephrons described by Oliver in chronic Bright's disease. The severely damaged anti-GBMN kidneys revealed widespread alterations in the proximal tubules, which, however, tended to be fairly uniform among the affected nephrons. This uniformity, perhaps, reflected the shorter duration of disease in the anti-GBMN animals. The most characteristic proximal tubular alteration in either the AICN or anti-GBMN animals was that combining atrophy of the pars convoluta and hypertrophy of the pars recta in same tubule. The largest and smallest nephrons encountered in the entire study were found in the most severly damaged kidney in the AICN. This finding reflected the simultaneous existence of regressive and progressive changes in this kidney, as certain nephrons underwent hypertrophy to compensate for the atrophy and disappearance of others. The remarkable functional glomerulotubular balance of single nephrons known to exist in both AICN and anti-GBMN was matched by the evidence presented here of structural glomerulotubular balance among the severely altered nephrons of a representative kidney from each group. Microdissection studies directed toward the characterization of the nephronic alterations in both membranous glomerulonephritis and proliferative glomerulonephritis of man should be of interest in relation to the findings in these experimental rat models of human disease.

Animals↗
PubMed1995

Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia.

The development and progression of human prostate cancer is associated with genetic abnormalities in tumor cells. Inactivation of tumor suppressor genes due to allelic loss is thought to be an important mechanism of gene alteration in prostatic neoplasms. In this study we examined allelic loss on chromosome 8p12-21 in microdissected samples of normal prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and invasive prostate carcinoma from the same patients. Tissue microdissection under direct microscopic visualization procures pure populations of cells of interest, including small lesions such as PIN. Among 30 patients with concomitant cancer and PIN, we found loss of heterozygosity on chromosome 8p12-21 in 63% (34 of 54) of foci of PIN examined and 90.6% (29 of 32) of tumors, suggesting that abnormalities on chromosome 8p12-21 may be important in the early stages of prostatic carcinoma development. Several cases in which multiple foci of PIN from the same patient were sampled showed different patterns of allelic loss. Fifty-five % (16 of 29) of the prostate carcinomas contained a potential precursor PIN focus based on allelic loss pattern. Our results are consistent with the hypothesis that PIN arises multifocally within the prostate gland, and that a subset of these lesions progress to become carcinoma.

Alleles↗